Figure 5. Bitopertin beneficially affects in vitro β-thalassemic erythropoiesis, with reduction of protoporphyrin IX, as a marker of heme restriction.

(A) Cell proliferation of β-thalassemic erythroid precursors derived by in vitro liquid culture of CD34⁺ cells isolated from peripheral blood of β-thal subjects with or without bitopertin 10 nM (n = 3). Arrows indicate when bitopertin 10 nM was added to the culture medium (see Supplemental Figure 6B for dose-response data on cell viability in normal erythropoiesis). Data are presented as mean ± SD; *P < 0.05 compared with vehicle-treated β-thal cells; Wilcoxon’s signed-rank test for multiple comparisons. (B) Effect of bitopertin (10 nM) on maturation pattern of β-thalassemic erythroid precursors from a single patient in triplicate. Representative flow cytometric plot and box plots of the differentiation pattern of β-thalassemic erythroid precursors at 14 days of culture with or without bitopertin (10 nM). The following surface markers were used: CD36, glycophorin-A, and CD71. This allows for the identification of the following homogenous cell populations: pro-erythroblasts (Pro-E), basophilic erythroblasts corresponding to intermediate erythroblasts (Int-E), and polychromatic and orthochromatic erythroblasts as late erythroblasts (Late-E) (see also refs. 4, 44). Data are shown as median and minimum/maximum (n = 4); *P < 0.05 compared with vehicle-treated β-thalassemic cells; Wilcoxon’s signed-rank test for multiple comparisons. (C) Effect of bitopertin on the index of erythroid maturation of β-thalassemic cells from 4 separate individuals at 9 and 14 days of cell culture. *P < 0.05 compared with vehicle-treated β-thalassemic cells; Mann-Whitney U test with multiple-comparisons correction. (D) Effect of bitopertin (10 nM) on the amount of annexin V⁺ cells during β-thalassemic erythroid maturation from 4 separate individuals at 9 and 14 days of cell culture. *P < 0.05 compared with vehicle-treated β-thalassemic cells, Mann-Whitney U test with multiple-comparison correction. (E) Intracellular content of protoporphyrin IX in healthy and β-thalassemic cells (4.5 × 10⁶ cells) after 14 days of cell cultures with and without bitopertin (10 nM). Data are presented as median and minimum/maximum (n = 4) from each group; *P < 0.05 compared with vehicle; Mann-Whitney U test with multiple-comparisons correction.